1) The study evaluated the effect of prolonged high-dose tibolone treatment on the vaginas of ovariectomized rats. 2) Histological analysis found the tibolone group had significantly thicker vaginal epithelium, more blood vessels, mast cells, and proliferating cells compared to the control group. 3) The results suggest high-dose long-term tibolone reverses vaginal atrophy in ovariectomized rats without causing dysplasia or tumors in the vagina.

1. Effect of prolonged use of high dose of tibolone on the vagina of ovariectomized rats
Helene Nara Henriques , *
Ana Carolina Bergmann de Carvalho , *
Porphirio José Soares Filho , *
José
Augusto Soares Pantaleão , †
and Maria Angélica Guzmán-Silva *
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Abstract
The aim of this study was evaluate the effect of prolonged use of high dose of tibolone on the
vagina of ovariectomized rats. Bilateral ovariectomy was performed on 14 rats weighing 250
g. Thirty days later, vaginal smears were collected verifying the menopause status by anoestrus
cytology. Rats were divided randomly into groups: experimental rats ( n = 9) received 1 mg
tibolone/day orally and control rats ( n = 6) received placebo (carboxymethylcellulose). After 150
days, all rats were sedated and euthanized by cervical displacement. The vagina was removed,
fixed in 10% buffered formalin, sampled and processed for paraffin embedding. Histological
sections were stained with haematoxylin and eosin, picrosirius red, periodic acid Schiff (PAS) and
PAS-diastase, and Weigert's resorcin–fuchsin. Cell proliferation was analysed by
immunohistochemistry to detect Ki67. Histomorphometric analyses were performed for epithelial
thickness, per cent area of collagen fibres and blood vessels, mast cells and Ki67-positive nuclei
per mm of basal membrane. Means and standard error of means were calculated, and data were
compared using the Mann–Whitney test, with significance level at P < 0.05. In the vagina,
epithelial thickness, number of Ki67-positive nuclei per mm of basal membrane, number of vessels
and number of mast cells were significantly higher in the tibolone group when compared with the
control group. Furthermore, the content of glycogen and glycoproteins in the vaginal epithelium
was modified by tibolone. Tibolone administered in high dose and for a long period has a trophic
effect, reversing vaginal atrophy, and has no dysplastic or neoplastic effect in the vagina of
ovariectomized rats.
Keywords: Ki67, menopause, ovariectomy, rats, tibolone, vagina
The decrease in circulating oestrogen observed in climacterium is responsible for a variety of
symptoms such as hot flushes, night sweats and vaginal atrophy.Approximately 75–80% of women
experience menopausal symptoms, and among them, 20–30% have severe symptoms (
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There is substantial evidence that the benefits of hormonal replacement therapy (HRT) in
menopause include the reduction in distressing symptoms as well as the reduction in the risk of
osteoporotic fractures, dementia and colorectal cancer, which improve well-being, quality of life,
sexual enjoyment and bladder capacity.
Material and methods
Animals

2. Fourteen Wistar rats, aged 8 weeks and weighing in average 250 g, were used. All rats were
produced and maintained in the Laboratory of Experimental Nutrition (LABNE) of the Fluminense
Federal University (UFF). Rats were housed in individual plastic cages, with controlled temperature
(24 ± 2 °C) and artificial illumination alternated in cycles of 12/12 h. Filtered water and commercial
food (Fri-Lab Ratos II, Fri-Ribe) were supplied ad libitum .
Ovariectomy
Bilateral ovariectomy was performed in all rats 30 days before the beginning of the experiment,
following the norms of vivisection of animals recommended by the Brazilian School of Animal
Experimentation (COBEA). The work was approved by the Committee of Ethics in Research of the
College of Medicine/Antônio Pedro Universitary Hospital/Fluminense Federal
University. Anaesthesia was intramuscular with 100 mg/kg of ketamine (Crystal) and 20 mg/kg of
xylazine (Anasedan Vetbrands) (
Chemicals
Tibolone was diluted at 0.2% in a solution of 0.5% carboxymethylcellulose (CMC).
Experimental design
After surgery, the rats did not receive medication for 30 days and received only food and water ad
libitum to reduce sex hormone levels and arrive at surgical menopause (
The rats were randomly distributed into two groups: The experimental group ( n = 9) received 0.5
ml per rat of tibolone, given 1 mg/day per rat. The control group ( n = 5) received daily doses of 0.5
ml per rat of CMC. Each group received their treatment by gavage administration for 150
consecutive days.
Vaginal smears were obtained immediately before the ovariectomy (day 30) to ensure that the
rats were in normal oestrus cycle. Thirty days after surgery (day 0), new vaginal cytology was
performed to verify the menopause status. After starting the administration of tibolone and CMC,
vaginal smears were collected on days 1 to 6, 30, 60, 90, 120 and 150 of the experiment to
evaluate the vaginal tropism, which is classified in oestrus, pro-oestrus, metoestrus and
dioestrus. Smears in oestrus, pro-oestrus and metoestrus indicate hormonal influence, and the
vaginal cytology in anoestrus points to a lack of hormonal influence and to vaginal atrophy. Smears
were immediately fixed in 95% alcohol and stained by the Papanicolaou method.
At the end of the experiment, all rats were anaesthetized with ketamine and xylazine and
euthanized by cervical dislocation. Vaginal tissues were removed, immediately fixed in 10%
buffered formalin, cleaved after 48 h and processed for paraffin embedding.
Histopathology
Histological sections of 4 μm obtained from the vagina were stained with haematoxylin and eosin
(HE). The histochemical reactions of periodic acid Schiff (PAS) and PAS-diastase were performed to
detect glycogen and glycoprotein respectively. Elastic fibres were analysed by the Weigert's
resorcin–fuchsin stain; muscle fibres and collagen were detected by the picrosirius red (PR)
stain. The collagen fibres were analysed using bright-field microscopy and circularly polarized light

3. microscopy to identify collagen fibres type I (orange birefringence) and collagen type III (green
birefringence).
Immunohistochemistry (IHC) for Ki67
Paraffin sections of 4 μm were deparaffinized in xylene and hydrated through graded alcohol
concentrations. The sections were pretreated with hydrogen peroxide to abolish endogenous
peroxidase activity and boiled in 10 mM citrate buffer at pH 6.0 in DakoCytomation Pascal
pressure chamber (DAKO, Carpinteria, CA, USA) pot for 45 min. Unspecific binding was blocked in
1% milk (Molico®, Nestlé Brasil Ltda., Barra Mansa, RJ, Brazil) and 1% bovine albumin for 15 min at
room temperature. The sections were incubated successively first with rabbit monoclonal anti-
ki67 antibody [diluted 1:100; (SP6) ab1667 Abcam®, Cambridge, MA, USA] for 1 h at room
temperature and later with secondary antibody and Streptavidin-Avidin-Biotin [LSAB®, 2 System-
HRP DakoCytomation K0609 (DAKO)] for 30 min at room temperature. The immunoreaction was
developed with 3,3′ -diaminobenzidine [DAB; DAKO liquid DAB + substrate chromogen system
K3467, (DAKO)], and the sections were lightly counterstained with haematoxylin. Sections of
human mammary carcinoma were used as positive controls.
HistomorphometryDigitized images of histological sections obtained under final magnification of
×400 were analysed using the I MAGE- P RO P LUS 4.5 software (Media Cybernetics, Silver Spring,
MD, USA).
Epithelial thicknessThe epithelial thickness was performed in slides stained with PR using the
drag-line tool in the image analysis program. Four images were taken from each animal, and the
average thickness was calculated from five measurements of each image.
Per cent area of collagen fibres and blood vesselsEight images from each animal were obtained,
and a 100-point grid mask was applied on each one. The manual count of structures was
performed, and the average per cent area of collagen fibres and blood vessels was determined per
group.
Mast CellsThe mast cells were counted under light microscopy at a final magnification of ×400 in
the vaginal submucosa in slides stained with PR. The average of mast cells per field was obtained
from the count in 10 microscopic fields for each animal.
Ki67-positive cellsTen images of vaginal epithelium were obtained from each animal for the
analysis of Ki67 immunohistochemistry positivity. A line was traced on the basal membrane to
determine its length (μm) in every image. The Ki67-positive nuclei were counted manually, and the
results were expressed as number of Ki67-positive nuclei per mm of basal membrane.
Statistical analysisResults are presented as mean and standard error of the mean (SEM). The
Shapiro–Wilk test was used to asses the normal distribution. Comparison between the groups was
made by the Mann–Whitney test. The G RAPH- P AD P RISM 5.0 statistical package (GraphPad
Software Inc., San Diego, CA, USA) was used for the analysis, and P < 0.05 was regarded as
statistically significant.
Results

4. At the beginning of the experiment, all the rats were in menopause confirmed by the vaginal
cytology similar to anoestrus. This pattern remained until the end of the experiment in the control
group. The oestrogenic effect of tibolone in the vagina was already observed at day 3 in the
tibolone group, because all rats had cytology similar to oestrus with cornified epithelial cells.
Epithelial morphology and histomorphometry
In control animals, there was a significant thinning of the squamous epithelium consisting of
approximately one to four layers of cells. On the other hand, in tibolone-treated animals, the
squamous epithelium presented approximately 10 cellular layers (Figure 1a,b ). These findings
were confirmed by histomorphometric analysis showing a significant increase in tibolone epithelial
thickness when compared with control group (75.06 ± 4.40 μm vs. 7.32 ± 0.64 μm, P <
0.001; Figure 2a ). Epithelial infoldings into the lamina propria were observed in the tibolone
group. The basal cells were columnar and there was a progressive flattening of cells towards the
luminal surface. Therefore, keratohyalin granules could be seen in the most superficial layers, and
cornification was noted in all tibolone-treated animals. Only sparse epithelial infoldings into
lamina propria were observed in control animals, and the cells showed very little surrounding
cytoplasm. Consistent with the decreased number of cell layers, there was no gradual transition to
anucleated squamous cells at the epithelial surface; the basal cells were cuboidal and when more
than a single epithelial cell layer was present, the superficial layer contained flattened cells
without keratohyalin granules.
Submucosal morphology and histomorphometry
No difference in morphology of the collagen and elastic fibres was registered between the
groups. Predominance of type I over type III collagen fibres was observed in both tibolone and
control groups. Moreover, similar dense disposition of collagen fibres was seen. This result was
confirmed by histomorphometry, which showed no significant difference in the per cent area of
collagen fibres between the tibolone and control groups (71.63 ± 2.47 vs. 76.20 ± 3.69; Figure
2b ). Sparse and thin elastic fibres in both groups were seen, corresponding to oxytalan fibres
responsible for tissue resistance. The per cent area of blood vessels and the mast cells count was
significantly higher in the tibolone group when compared with the control group (22.05 ±
2.33 vs.12.15 ± 1.12, P < 0.01 and 1.27 ± 0.16 vs. 0.44 ± 0.12, P < 0.05 respectively; Figure 2c,d ).
Cellular proliferation
Frequent mitotic figures were present in the basal layer of tibolone group. On the other hand,
these figures were not seen in the control group. Consistent with these findings, the number of
Ki67-positive nuclei per mm of basal membrane was significantly higher in the tibolone group
when compared with the control group (57.95 ± 8.47 vs. 9.77 ± 6.32, P < 0.01; Figure 2e ).
Glycogen and glycoprotein detection
Glycogen was detected in all animals from tibolone group by the PAS-positive and PAS-diastase-
negative reaction. PAS positivity was seen in intraepithelial leucocytes and superficial epithelial
cells with keratohyalin granules ( Figure 1c,d ). Glycoprotein was observed in all rats from the
control group by the positive reactions of PAS and PAS-diastase.

5. Discussion
The high dose of tibolone used in this experiment was similar to other studies (
studying the effects of the drug on the bone tissue in short-term (4 weeks) and long-term (64
weeks) treatments. One hundred and fifty days of treatment represent a prolonged period as it
corresponds approximately to one sixth of an expected lifetime of a rat. The extension of our
experimental protocol according to the ICH and OECD guidelines for chronic toxicity and
carcinogenicity studies ( was between subacute (2–13 weeks) and chronic (24–104 weeks) studies
and was carried out to clarify possible toxicity the liver (data not shown) and potential deleterious
effects on target organs by continuous exposure to tibolone The initial data clearly show the
oestrogen effect of tibolone on the vagina evidenced by vaginal cytology showing only cornified
epithelial cells similar to the oestrus phase.On the other hand, the vaginal smears of control group
confirmed the lack of hormonal action because of surgical menopause. The Allen–Doisy vaginal
cornification assay is used almost exclusively for the biological assay of oestrogenic compounds in
vaginal smears assessed according to their cellular composition, the simplest method of scoring
being to record those with cornified or nucleated epithelial cells but no leucocytes as positive and
the rest as negative. We used other validated and frequently used methods in which the various
stages of the oestrus cycle were recorded.
In this study, we investigated the effects of tibolone treatment on the vaginal tissue architecture
of ovariectomized rats. Our results showed that ovariectomy exerted a powerful effect on the
vaginal epithelium leading to intense atrophy. This drastic structural alteration is related to
changes in the functional properties of the epithelium, which becomes more susceptible to
abrasion, supporting clinical findings in which the epithelium is reported to be more fragile after
menopause ( A lower protection against bacterial infection in women during menopause may be
owing to thinner epithelium and loss of its ability to produce glycogen. This can lead to changes in
the vaginal flora composition, increasing the risk of infections on the lower urinary tract. The
tibolone treatment reversed most changes in tissue rat morphology noted in this study after
ovariectomy. The epithelium was remarkably hyperplasic and recovered its ability to produce
glycogen.The glycogen present in the vaginal epithelium of tibolone group probably leads to
vaginal flora recovery as well as occurs with women using HRT ( Ovarian hormones seem to be
regulatory factors that favour the presence of a broad variety of bacteria, which are members of
the normal genital tract flora. On the other hand, ovariectomy modifies the vaginal microbial
profile, and hormone replacement therapy alleviates this disturbance.
After the tibolone treatment, our results were similar to the effect of hormone administration
(oestradiol, oestradiol + testosterone and oestradiol + progesterone), which restored the epithelial
thickness to what was seen before the ovariectomy in rats; the vaginal stratified squamous
epithelium consisted of approximately the same number of layers seen in the non-ovariectomized
rats. Tibolone had no influence on per cent area of collagen fibres in rats as well as in collagen
type I/III ratio and the per cent area of elastic fibres. The oestrogen treatment in women leads to a
decrease in the collagen content when compared with placebo treatment; however, there is no
effect in the collagen type I/III ratio (Thus, tibolone appears to exert a weak oestrogen effect on
the vaginal collagen content of ovariectomized rats. The reduction of plasma oestradiol observed
in diabetic mice leads to changes consistent with the menopause condition in the vagina, such as
reduced epithelial thickness, reduced muscle layer and change in the arrangement of elastic
fibres. Oestradiol administration significantly increases the volume of the muscle layer and the
lamina propria, but induces less marked changes in elastic fibres (

6. Also, no remarkable changes were noted in the vaginal elastic fibres by the tibolone treatment.
The per cent area of blood vessels on the vaginal submucosa was higher in the tibolone group
when compared with the control group. The oestrogen replacement in rats leads to an increase in
the blood flow, which could be explained by the increase in per cent area of blood vessels found in
this study.
The cellular proliferation – regenerative hyperplasia – was higher in the tibolone group when
compared with the control rats. This result was evidenced by histopathology and
histomorphometry. Data were comparable to a study in women showing that the Ki67 expression
was higher in the oestrogen-treated group when compared with non-treated postmenopausal
Mast cells express high affinity for oestrogen receptors, and their granule secretion is increased by
This fact was proven by the study performed in possums ( Trichosurus vulpecula ), which showed
an inverse relation between the mast cells number in the vagina and oestrogen. However, in the
present study, an increase in non-degranulating mast cells was observed in the vaginal submucosa
of the tibolone group when compared with the control group. In contrast, tibolone metabolites
appeared not to be able to trigger degranulation of mast cells, which could explain why an
increased number of these cells was observed in the vagina of tibolone-treated rats.
Tibolone administered in high dosages and for a long period of time has proliferative and trophic
effects, regenerative hyperplasia, reversing vaginal atrophy, and does not have dysplastic or
neoplastic effects in the vagina of ovariectomized rats. However, a very careful extrapolation of
these results to humans is necessary as the background clinical history may exert influence of the
response to tibolone hormonal therapy.